Message-Id: <s2ccee38.098@genetics.com>
Date: Fri, 03 Jan 1997 11:37:50 -0500
From: Elliott Nickbarg <ENickbarg@genetics.com>
Subject: High Sensitivity MS/MS Sequencing -Reply
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Sub-picomole level peptide sequencing with MS/MS technology has
been achievable now for several years. Several years ago, my lab, a
protein chemistry department in a biotech company, and which had no
experience with ms, decided that we needed to learn to do ms/ms
sequencing in order to do high-sensivity sequencing and structural
analysis of proteins and peptides. Since we are always
sample-limited, the (potentially) high sensitivity of ms was very
attractive. We bought a triple quad and leaned how to deal with ms
and ms/ms. As has been shown by a number of investigators, MS is
definitely very complementary to Edman, and can be applied to a whole
range of structural problems. We have solved problems that would not
have worked with Edman, and the demands on the instruments (and
ourselves) keep increasing. Because of their potentially high
throughput, I think that the value of ms will be greater in the
post-genome world.
Now for the bad news. The problem with saying that one can
sequence 1 fmol (or whatever) with ms, is that it is a bit like
saying that a 747 jet can stop on a dime--if the dime is placed at
the end of a long runway. We needed to devote a couple of people to
learn new data analysis techniques and invest time in adapting and
developing sample handling techniques. The newest generation of ms
instruments is easier to use, but there is no question that knowledge
is required to get the sample safely into the instrument and to
interpret the results afterward. Specialized sample handing
techniques (micro-capillary chromatography, nanospray, CE-ms, to name
a few) can all routinely yield good data with low-fmol amounts of
sample, but one needs experience to use these methods routinely and
to evaluate which method to use for a given sample. If you want to
push the ms (either maldi or esi) to its highest possible
sensitivity, you will need to devote time to learning the appropriate
sample handling tehniques.
The time needed for data interpretation also is significant.
While new computer algorithms have been to developed to screen ms/ms
data against sequence databases (Yates' Sequest and Mann's
Peptidesearch), experience is still necessary to properly evaluate
results. These programs make high-throughput ms/ms possible.
In summary--MS can become integrated into a core lab if one has a
motivated person or two to focus their complete effort on it. I
think that ms is definitely worth this investment.
Elliott Nickbarg
(As usual, the opinions I express are those of myself, and not of my
company, etc, etc...)
Genetics Inst. Inc.
Protein Biochemistry